Genome mining conformance to metabolite profile of Bacillus strains to control potato pathogens.

Biocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp., responsible for potato soft rot disease. Discovering agents that can effectively control both fungal and bacterial pathogens in potatoes has always presented a challenge. Biological controls were investigated using 500 bacterial strains isolated from rhizospheric microbial communities, along with two promising biocontrol strains: Pseudomonas (T17-4 and VUPf5). Bacillus velezensis (Q12 and US1) and Pseudomonas chlororaphis VUPf5 exhibited the highest inhibition of fungal growth and pathogenicity in both laboratory (48%, 48%, 38%) and greenhouse (100%, 85%, 90%) settings. Q12 demonstrated better control against bacterial pathogens in vivo (approximately 50%). Whole-genome sequencing of Q12 and US1 revealed a genome size of approximately 4.1 Mb. Q12 had 4413 gene IDs and 4300 coding sequences, while US1 had 4369 gene IDs and 4255 coding sequences. Q12 exhibited a higher number of genes classified under functional subcategories related to stress response, cell wall, capsule, levansucrase synthesis, and polysaccharide metabolism. Both Q12 and US1 contained eleven secondary metabolite gene clusters as identified by the antiSMASH and RAST servers. Notably, Q12 possessed the antibacterial locillomycin and iturin A gene clusters, which were absent in US1. This genetic information suggests that Q12 may have a more pronounced control over bacterial pathogens compared to US1. Metabolic profiling of the superior strains, as determined by LC/MS/MS, validated our genetic findings. The investigated strains produced compounds such as iturin A, bacillomycin D, surfactin, fengycin, phenazine derivatives, etc. These compounds reduced spore production and caused deformation of the hyphae in F. solani. In contrast, B. velezensis UR1, which lacked the production of surfactin, fengycin, and iturin, did not affect these structures and failed to inhibit the growth of any pathogens. Our findings suggest that locillomycin and iturin A may contribute to the enhanced control of bacterial pectolytic rot by Q12.

Microencapsulation of a Pseudomonas Strain (VUPF506) in Alginate-Whey Protein-Carbon Nanotubes and Next-Generation Sequencing Identification of This Strain.

Alginate is a common agent used for microencapsulation; however, the formed capsule is easily damaged. Therefore, alginate requires blending with other biopolymers to reduce capsule vulnerability. Whey protein is one polymer that can be incorporated with alginate to improve microcapsule structure. In this study, three different encapsulation methods (extrusion, emulsification, and spray drying) were tested for their ability to stabilize microencapsulated Pseudomonas strain VUPF506. Extrusion and emulsification methods enhanced encapsulation efficiency by up to 80% and gave the best release patterns over two months. A greenhouse experiment using potato plants treated with alginate-whey protein microcapsules showed a decrease in Rhizoctonia disease intensity of up to 70%. This is because whey protein is rich in amino acids and can serve as a resistance induction agent for the plant. In this study, the use of CNT in the ALG-WP system increased the rooting and proliferation and reduced physiological complication. The results of this study showed that the technique used in encapsulation could have a significant effect on the efficiency and persistence of probiotic bacteria. Whole genome sequence analysis of strain VUPF506 identified it as Pseudomonas chlororaphis and revealed some genes that control pathogens.

Gibbsiella quercinecans as new pathogen involved in bacterial canker of Russian olive.

Russian olive (Elaeagnus angustifolia) is an economically important ornamental and crop plant. It has a wide range of biologically active compounds and is regarded as a unique medicinal plant with multiple applications. Members of the order Enterobacteriales isolated from trees are often associated with bacterial canker. During the growing season from 2017 to 2020, forty Gram-negative bacterial strains were isolated from Russian olive trees infected with cankers showing symptoms such as distinctive lesions on the trunk and branches, decline, weakness of trees, cracked bark, depressed brown to black lesions accompanied by exudation of gum, in the Kerman and Mazandaran Provinces of Iran. We used a polyphasic approach to identify and characterize these strains. Using a multilocus sequence analysis approach of four housekeeping loci, namely atpD, rpoB, gyrB, infB, and partial 16S rRNA gene sequences, isolates were identified as Gibbsiella quercinecans. These results were further supported by phenotypic and biochemical tests. Results of the biochemical, physiological and phenotypic experiments, indicated that the isolates are members of the Enterobacteriales and within the genus Gibbsiella. Pathogenicity of the G. quercinecans isolates was confirmed by fulfillment of Koch's postulates. To our knowledge, this is the first report of G. quercinecans as the causal agent of bacterial canker of Russian olive trees.

Genomics-Enabled Novel Insight Into the Pathovar-Specific Population Structure of the Bacterial Leaf Streak Pathogen Xanthomonas translucens in Small Grain Cereals.

The Gram-negative bacterium Xanthomonas translucens infects a wide range of gramineous plants with a notable impact on small grain cereals. However, genomics-informed intra-species population structure and virulence repertories of the pathogen have rarely been investigated. In this study, the complete genome sequences of seven X. translucens strains representing an entire set of genetic diversity of two pathovars X. translucens pv. undulosa and X. translucens pv. translucens is provided and compared with those of seven publicly available complete genomes of the pathogen. Organization of the 25 type III secretion system genes in all the 14 X. translucens strains was exactly the same, while TAL effector genes localized singly or in clusters across four loci in X. translucens pv. translucens and five to six loci in X. translucens pv. undulosa. Beside two previously unreported endogenous plasmids in X. translucens pv. undulosa, and variations in repeat variable diresidue (RVD) of the 14 strains, tal1a of X. translucens pv. translucens strain XtKm8 encode the new RVDs HE and YI which have not previously been reported in xanthomonads. Further, a number of truncated tal genes were predicted among the 14 genomes lacking conserved BamHI site at N-terminus and SphI site at C-terminus. Our data have doubled the number of complete genomes of X. translucens clarifying the population structure and genomics of the pathogen to pave the way in the small grain cereals industry for disease resistance breeding in the 21st century's agriculture.

Survivability and controlled release of alginate-microencapsulated Pseudomonas fluorescens VUPF506 and their effects on biocontrol of Rhizoctonia solani on potato.

Preserving the efficacy of plant probiotic bacteria in soil is a major challenge to the biological control of plant diseases. The microencapsulation technique is an important step in preserving the viability and activity of probiotics in adverse environmental conditions. The main objective of this study was to choose an appropriate coating for probiotic encapsulation. For this purpose, the survivability and controlled release of Pseudomonas fluorescens VUPF506 encapsulated with alginate (Alg) combined with whey protein concentrate (WPC), carboxymethyl cellulose (CMC), and peanut butter (PB) were evaluated. Moreover, the encapsulated cells were evaluated to control for Rhizoctonia solani in potato plants under in vivo conditions. The results showed that all tested wall material maintained more than 80% of the bacterial cells. The Alg-WPC microcapsules provided a better controlled release over two months. Interestingly, the greenhouse experiment also revealed that the treatment of potato plants with Alg-WPC microcapsules was the most effective treatment, suppressing 90% of the pathogen. The results showed that Alg-WPC is the most promising combination to improve the survivability of P. fluorescens VUPF506. Moreover, it can be used as a fertilizer due to its content of valuable amino acids.

Biocontrol potential of native yeast strains against Aspergillus flavus and aflatoxin production in pistachio.

ASPERGILLUS FLAVUS: is the main aflatoxin producer in food and feed and has wide ecological niches. Contamination of food products such as pistachio nuts and aflatoxin secretion directly affects food safety and international food product trades. Abilities of 13 yeast strains isolated from 200 soil and pistachio nut samples collected in Iranian orchards to reduce the growth of A. flavus as well as aflatoxin production were assessed in dual culture, volatile and non-volatile compounds tests. The growth of A. flavus was reduced by 32-60%, 13-31% and 40-61% in dual culture, volatile and non-volatile compounds, respectively, while aflatoxin B1 production was diminished by 90.6-98.3%. Based on these assays, five yeast strains were selected for co-inoculation experiments using soil, pistachio hulls and leaf. A significant reduction in colony-forming units (CFU) ranging from 23% to 110% (p < .05) was observed. Molecular, physiological and morphological identification revealed these were strains of Pichia kudriavzevii and Lachansea thermotolerans. Aflatoxin biocontrol with yeast strains possesses many advantages including the ease of commercial production and organic application which is an environmental approach. More investigation is required to understand the efficiency of selective strains to inhibit A. flavus and aflatoxin production as well as withstand predominant abiotic stress in pistachio orchards and mass production in field application.

Molecular Typing Reveals High Genetic Diversity of Xanthomonas translucens Strains Infecting Small-Grain Cereals in Iran.

This study provides a phylogeographic insight into the population diversity of Xanthomonas translucens strains causing bacterial leaf streak disease of small-grain cereals in Iran. Among the 65 bacterial strains isolated from wheat, barley, and gramineous weeds in eight Iranian provinces, multilocus sequence analysis and typing (MLSA and MLST) of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD), identified 57 strains as X. translucens pv. undulosa, while eight strains were identified as X. translucens pv. translucens. Although the pathogenicity patterns on oat and ryegrass weed species varied among the strains, all X. translucens pv. undulosa strains were pathogenic on barley, Harding's grass, rye (except for XtKm35) and wheat, and all X. translucens pv. translucens strains were pathogenic on barley and Harding's grass, while none of the latter group was pathogenic on rye or wheat (except for XtKm18). MLST using the 65 strains isolated in Iran, as well as the sequences of the four genes from 112 strains of worldwide origin retrieved from the GenBank database, revealed higher genetic diversity (i.e., haplotype frequency, haplotype diversity, and percentage of polymorphic sites) among the Iranian population of X. translucens than among the North American strains of the pathogen. High genetic diversity of the BLS pathogen in Iran was in congruence with the fact that the Iranian Plateau is considered the center of origin of cultivated wheat. However, further studies using larger collections of strains are warranted to precisely elucidate the global population diversity and center of origin of the pathogen.IMPORTANCE Bacterial leaf streak (BLS) of small-grain cereals (i.e., wheat and barley) is one of the economically important diseases of gramineous crops worldwide. The disease occurs in many countries across the globe, with particular importance in regions characterized by high levels of precipitation. Two genetically distinct xanthomonads-namely, Xanthomonas translucens pv. undulosa and X. translucens pv. translucens-have been reported to cause BLS disease on small-grain cereals. As seed-borne pathogens, the causal agents are included in the A2 list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Despite its global distribution and high economic importance, the population structure, genetic diversity, and phylogeography of X. translucens remain undetermined. This study, using MLSA and MLST, provides a global-scale phylogeography of X. translucens strains infecting small-grain cereals. Based on the diversity parameters, neutrality indices, and population structure, we observe higher genetic diversity of the BLS pathogen in Iran, which is geographically close to the center of origin of common wheat, than has so far been observed in other areas of the world, including North America. The results obtained in this study provide a novel insight into the genetic diversity and population structure of the BLS pathogen of small-grain cereals on a global scale.

Effect of Arsenophonus Endosymbiont Elimination on Fitness of the Date Palm Hopper, Ommatissus lybicus (Hemiptera: Tropiduchidae).

The date palm hopper, Ommatissus lybicus de Bergevin, is one of the most important pests of the date palm in the Middle East and North Africa. This insect uses its needle-like sucking mouthparts to feed on phloem, which is devoid of most essential amino acids and many vitamins. The absence of essential nutrient in its diet is suggested to be ameliorated by endosymbionts in O. lybicus. Arsenophonus is one of the main bacterial endosymbionts widely prevalent in O. lybicus. In this study, we used antibiotics to eliminate Arsenophonus from O. lybicus originating from three populations (Fin, Qale'e Qazi, and Roodan) and studied the effects on the fitness of the pest. Our results revealed that the removal of Arsenophonus increased the developmental time of the immature stages and reduced the values of different life-history parameters including nymphal survival rate and adult longevity in the host. Furthermore, elimination of Arsenophonus completely obliterated offspring production in all O. lybicus populations investigated. These results confirm the dependency of O. lybicus on Arsenophonus for fitness and give a new insight regarding the possibility of symbiotic control of O. lybicus.

Screening bactericidal effect of Pectobacterium carotovorum subsp. carotovorum strains against causal agent of potato soft rot.

This study focuses on the potential of Pectobacterium carotovorum subsp. carotovorum (Pcc) strains producing bacteriocin as a tool to control potato soft rot disease. Thirty out of 48 purified bacterial strains were characterized as Pcc using specific PCR and phenotypic tests. The pathogenicity and pectate degrading assays were recorded positive for 13 strains. Bacteriocin typing clustered producers into three groups according to their antimicrobial spectra. Majority of the producers except strains of group II showed antibacterial activity toward relative genus and the role of UV or mitomycin C was inductive. In addition, none of the distant genus was sensitive to Pcc bacteriocins except Rhizobium vitis. Molecular detection of four bacteriocins including carotovoricin, carosin S1, S2 and carosin D was performed. Overall, 54.5% of group I, 47.3 and 70% of groups II and III strains carried carotovoricin and four strains harbored gene corresponding to carosin S1. According to our data divers antimicrobial patterns obtained by Pcc strains and existence of new bateriocines could be possible. Moreover, our findings recommended that direct application of P29 or expression of corresponding genes of Pog22 or P21 in a nonpathogenic strain as a biocontrol agent may improve soft rot disease control.