Inhibition of the Citrus Bacterial Pathogen Xanthomonas axonopodis pv. citri through Aloysia gratissima Essential Oil and Flourensia spp. Extracts and Prenylflavonoids

Background: Xanthomonas axonopodis pv. citri is a gram-negative bacterium that affects citrus crops, causing a disease known as citrus canker. Although essential oils and other compounds isolated from plants represent a natural alternative to treat this disease, they have the disadvantage of having low solubility in the media in which the bioassays to determine antimicrobial activity are performed. This has led several researchers to evaluate the solubility of plant essential oils in alternative solvents. Objectives: The aim of this study was to evaluate the solubility of the essential oil from Aloysia gratissima as well as that of low-polarity extracts and pure compounds of the genus Flourensia in diluted agar/Tween 80 solutions to test and improve their antimicrobial activity against Xanthomonas axonopodis pv. citri. Methods: Antimicrobial activity against Xanthomonas axonopodis pv. citri was determined by bioautography, agar diffusion, and microdilution methods. Results: The A. gratissima oil showed increased activity in the agar (0.15 % m/v)/Tween80 (0.5 % v/v) 1:1 mixture, with MIC values ranging from 75 to 100 µL/mL, while Flourensia spp. extracts were more soluble in agar solution (0.15 % m/v). The pure compounds tested presented MIC values ranging from 50 to 150 µg/mL. Conclusion: The proven antimicrobial activity of both Aloysia gratissima essential oil and Flourensia spp. extracts and pure compounds allows proposing these natural products as potential antimicrobial agents in the control of citrus canker.

Analysis of CcGASA family members in Citrus clementina (Hort. ex Tan.) by a genome-wide approach

The Gibberellic Acid Stimulated Arabidopsis (GASA) proteins were investigated in the study to help understand their possible roles in fruit trees, particularly in Citrus. A total of 18 CcGASA proteins were identified and characterized in Citrus clementina via a genome-wide approach. It was shown that the CcGASA proteins structurally shared a conserved GASA domain but varied considerably in primary sequences and motif compositions. Thus, they could be classified into three major phylogenetic groups, G1~G3, and two groups, G1 and G3 could be further classified into subgroups. The cis- elements on all CcGASA promoters were identified and categorized, and the associated transcription factors were predicted. In addition, the possible interactions between the CcGASA proteins and other proteins were predicted. All the clues suggested that these genes should be involved in defense against biotic and abiotic stresses and in growth and development. The notion was further supported by gene expression analysis that showed these genes were more or less responsive to the treatments of plant hormones (GA3, SA, ABA and IAA), and infections of citrus canker pathogen Xanthomonas citri. It was noted that both the segmental and the tandem duplications had played a role in the expansion of the CcGASA gene family in Citrus. Our results showed that the members of the CcGASA gene family should have structurally and functionally diverged to different degrees, and hence, the representative group members should be individually investigated to dissect their specific roles.

Comparative Genomic Analysis Uncovered Evolution of Pathogenicity Factors, Horizontal Gene Transfer Events, and Heavy Metal Resistance Traits in Citrus Canker Bacterium Xanthomonas citri subsp. citri

Background: Worldwide citrus production is severely threatened by Asiatic citrus canker which is caused by the proteobacterium Xanthomonas citri subsp. citri. Foliar sprays of copper-based bactericides are frequently used to control plant bacterial diseases. Despite the sequencing of many X. citri strains, the genome diversity and distribution of genes responsible for metal resistance in X. citri subsp. citri strains from orchards with different management practices in Taiwan are not well understood.Results: The genomes of three X. citri subsp. citri strains including one copper-resistant strain collected from farms with different management regimes in Taiwan were sequenced by Illumina and Nanopore sequencing and assembled into complete circular chromosomes and plasmids. CRISPR spoligotyping and phylogenomic analysis indicated that the three strains were located in the same phylogenetic lineages and shared ∼3,000 core-genes with published X. citri subsp. citri strains. These strains differed mainly in the CRISPR repeats and pathogenicity-related plasmid-borne transcription activator-like effector (TALE)-encoding pthA genes. The copper-resistant strain has a unique, large copper resistance plasmid due to an unusual ∼40 kbp inverted repeat. Each repeat contains a complete set of the gene cluster responsible for copper and heavy metal resistance. Conversely, the copper sensitive strains carry no metal resistance genes in the plasmid. Through comparative analysis, the origin and evolution of the metal resistance clusters was resolved.Conclusion: Chromosomes remained constant among three strains collected in Taiwan, but plasmids likely played an important role in maintaining pathogenicity and developing bacterial fitness in the field. The evolution of pathogenicity factors and horizontal gene transfer events were observed in the three strains. These data suggest that agricultural management practices could be a potential trigger for the evolution of citrus canker pathogens. The decrease in the number of CRISPR repeats and pthA genes might be the result of adaptation to a less stressful environment. The metal resistance genes in the copper resistant X. citri strain likely originated from the Mauritian strain not the local copper-resistant X. euvesicatoria strain. This study highlights the importance of plasmids as ‘vehicles’ for exchanging genetic elements between plant pathogenic bacteria and contributing to bacterial adaptation to the environment.

First report of Xanthomonas citri Subsp. Citri causing citrus canker on grape fruit (Citrus paradisi), washington naval (Citrus sinensis), kaghzi limon (Citrus aurantifolia Swingle), lemon (Citrus lim

Citrus fruit production is largely affected by different bacterial and fungal pathogens. In Pakistan bacterial diseases like citrus bacterial canker (CBC) pose severe risk to citrus economy. Diagnoses of such diseases could be helpful to avoid the epidemics in nurseries or orchids. In 2011-12, citrus canker symptoms i.e., callus-like outgrowths on leaves and fruits of grape fruit (Citrus paradisi), Washington naval (Citrus sinensis), Kaghzi Limon (Citrus aurantifolia swingle), lemon (Citrus Limon) and pomelo (Citrus maxima) were noticed in Sargodha district of Punjab, Pakistan. Bacteria i.e., yellow mucoid, Xanthomonas- like isolates, were isolated from these lesions. Bacteria isolated from these lesions were cultured and total DNA was isolated. A diagnostic fragment of 581 bp based on rpf genes of Xanthomonas citri pv. citri was amplified, cloned and completely sequenced. BLAST and evolutionary analysis revealed that these isolates show 100% sequence similarity and group with Xanthomonas citri subsp. citri from Argentina (CP023285) and Reunion (CP018858), (CP018854). To our knowledge, this is the first formal report of X. campestris pv. citri pathotypes A on Citrus paradise, Citrus sinensis, Citrus maxima, Citrus Limon and Citrus aurantifolia swingle in Pakistan

Potential Bioinoculants for Sustainable Agriculture Prospected from Ferruginous Caves of the Iron Quadrangle/Brazil

Biocontrol and plant growth-promoting bacteria (PGPB) are important agricultural bioinoculants. This study aimed to prospect new potential bioinoculants for a more sustainable agriculture from ferruginous caves of the Brazilian Iron Quadrangle. Culturable bacteria, from seven caves and one canga soil sample, were evaluated for biocontroller activity of the phytopathogens Xanthomonas citri subsp. Citri—Xcc306 (citrus canker), Fusarium oxysporum—Fo (fusariosis), and Colletotrichum lindemuthianum—Cl89 (bean anthracnose). The ability of the superior candidates to solubilize inorganic phosphate, fix nitrogen, and produce hydrolytic enzymes and siderophores was then analyzed. Out of 563 isolates, 47 inhibited the growth of Xcc306 in vitro, of which 9 reduced citrus canker up to 68% when co-inoculated with the pathogen on host plants. Twenty of the 47 inhibited Fo growth directly by 51–73%, and 15 indirectly by 75–81%. These 15 inhibited Cl89 growth in vitro (up to 93% directly and 100% indirectly), fixed nitrogen, produced proteases and siderophores, showed motility ability, produced biofilm, and all but one solubilized inorganic phosphate. Therefore, 15 (2.66%) bacterial isolates, from the genera Serratia, Nissabacter, and Dickeya, act simultaneously as biocontrollers and PGPBs, and could be important candidates for future investigations in planta as an alternative to minimize the use of pesticides and chemical fertilizers through sustainable agricultural management practices.