Evaluating the Antibacterial Activity and Mode of Action of Thymol-Loaded Chitosan Nanoparticles Against Plant Bacterial Pathogen Xanthomonas campestris pv. campestris

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in cruciferous crops, resulting in severe yield loss worldwide. The excessive use of chemical pesticides in agriculture to control diseases has raised significant concern about the impact on the environment and human health. Nanoparticles have recently gained significant attention in agriculture owing to their promising application in plant disease control, increasing soil fertility and nutrient availability. In the current study, we synthesized thymol-loaded chitosan nanoparticles (TCNPs) and assessed their antibacterial activity against Xcc. The synthesis of TCNPs was confirmed by using ultraviolet–visible spectroscopy. Fourier-transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy analysis revealed the functional groups, size, and shape of TCNPs, with sizes ranging from 54 to 250 nm, respectively. The antibacterial activity of TCNPs against Xcc was investigated in vitro by liquid broth, cell viability, and live dead staining assay, and all of them demonstrated the antibacterial activity of TCNPs. Furthermore, TCNPs were found to directly inhibit the growth of Xcc by suppressing the growth of biofilm formation and the production of exopolysaccharides and xanthomonadin. The ultrastructure studies revealed membrane damage in TCNP-treated Xcc cells, causing a release of intracellular contents. Headspace/gas chromatography (GC)–mass spectrometry (MS) analysis showed changes in the volatile profile of Xcc cells treated with TCNPs. Increased amounts of carbonyl components (mainly ketones) and production of new volatile metabolites were observed in Xcc cells incubated with TCNPs. Overall, this study reveals TCNPs as a promising antibacterial candidate against Xcc.

Identification of Pathogenic Fusarium spp. Responsible for Root Rot of Angelica sinensis and Characterization of their Biological Enemies in Dingxi, China

Root rot is a serious disease in plantations of A. sinensis, severely affecting yield and quality and threatening sustainable production. Fusarium isolates (n=32) were obtained from field samples of root rot tissue, leaves and infected soil. Isolates were identified by comparing the sequences of their internal transcribed spacer (ITS) region and translation elongation factor 1-ɑ (TEF-1ɑ) to sequences of known species in the NCBI-database. These Fusarium isolates include F. tricinctum (43.75%), F. equiseti (31.25%), F. solani (9.37%), F. oxysporum (6.25%), F. acuminatum (6.25%), and F. incarnatum (3.12%). For pathogenicity testing under greenhouse conditions, seven isolates were selected based on a phylogenetic analysis, including four strains of F. tricinctum and one strain each of F. solani, F. oxysporum, and F. acuminatum. The seven isolates were all pathogenic but differed in their ability to infect: the four F. tricinctum strains were capable pathogens causing root rot in A. sinensis at 100% incidence and the highly aggressive. Furthermore, the symptoms of root rot induced by those seven isolates were consistent with typical root rot cases in the field, but their disease severity varied. Observed histopathological preparations of F. tricinctum-infected seedlings and tissue-slides results showed this fungal species can penetrate epidermal cells and colonize the cortical cells where it induces necrosis and severe plasmolysis. Plate confrontation experiments showed that isolated rhizosphere bacteria inhibited the Fusarium pathogens that cause root rot in A. sinensis. Our results provide timely information for informing the use of biocontrol agents for suppression of root rot disease.

Debaryomyces hansenii, Stenotrophomonas rhizophila, and Ulvan as Biocontrol Agents of Fruit Rot Disease in Muskmelon (Cucumis melo L.)

The indiscriminate use of synthetic fungicides has led to negative impact to human health and to the environment. Thus, we investigated the effects of postharvest biocontrol treatment with Debaryomyces hansenii, Stenotrophomonas rhizophila, and a polysaccharide ulvan on fruit rot disease, storability, and antioxidant enzyme activity in muskmelon (Cucumis melo L. var. reticulatus). Each fruit was treated with (1) 1 × 106 cells mL−1 of D. hansenii, (2) 1 × 108 CFU mL−1 of S. rhizophila, (3) 5 g L−1 of ulvan, (4) 1 × 106 cells mL−1 of D. hansenii + 1 × 108 CFU mL−1 of S. rhizophila, (5) 1 × 108 CFU mL−1 of S. rhizophila + 5 g L−1 of ulvan, (6) 1 × 106 cells mL−1 of D. hansenii + 1 × 108 CFU mL−1 of S. rhizophila + 5 g L−1 of ulvan, (7) 1000 ppm of benomyl or sterile water (control). The fruits were air-dried for 2 h, and stored at 27 °C ± 1 °C and 85–90% relative humidity. The fruit rot disease was determined by estimating the disease incidence (%) and lesion diameter (mm), and the adhesion capacity of the biocontrol agents was observed via electron microscopy. Phytopathogen inoculation time before and after adding biocontrol agents were also recorded. Furthermore, the storability quality, weight loss (%), firmness (N), total soluble solids (%), and pH were quantified. The antioxidant enzymes including catalase, peroxidase, superoxide dismutase, and phenylalanine ammonium lyase were determined. In conclusion, the mixed treatment containing D. hansenii, S. rhizophila, and ulvan delayed fruit rot disease, preserved fruit quality, and increased antioxidant activity. The combined treatment is a promising and effective biological control method to promote the shelf life of harvested muskmelon.

Relationship of Resistance-Related Enzyme activity and Salicylic Acid content in Phalaenopsis Species with different levels of resistance to DIckeya Dadantii

ABSTRACT Orchids (Phalaenopsis) are ornamental plants that are cultivated commercially and in great demand in the market. Soft-rot disease (SRD) caused by the necrotrophic pathogen Dickeya dadantii is a cause of considerable economic loss to cultivators of many orchid species. Our previous experiment identified a limited number of species that were resistant to D. dadantii. This study aimed to validate the resistance level of four Phalaenopsis species in a detached leaf inoculation protocol to identify the resistance mechanism(s) involved. Soft-rot symptom diameter was measured from 6 to 18 hours post-inoculation (HPI) with D. dadantii. Disease assessment confirmed that P. amboinensis is a resistant species, P. pantherina is a susceptible species, and P. amabilis and P. schilleriana are very susceptible species. There was no difference in the lignin content between the resistant and very susceptible species. Detailed observation of resistant and very susceptible species, P. amboinensis vs. P. amabilis, revealed higher phenylalanine ammonia-lyase (PAL) and peroxidase (POD) in P. amabilis than in P. amboinensis. In contrast, there was higher salicylic acid (SA) content in P. amboinensis than in P. amabilis. These results suggest that POD and PAL activities may not be effective in defense against soft-rot disease, while SA plays an important role in the resistance of P. amboinensis to D. dadantii. Low PAL activity in P. amboinensis implies that the SA contents from the isochorismate pathway may be involved in the mechanism of P. amboinensis resistance to D. dadantii. Therefore, endogenous SA content may be a good indicator for screening resistant species in Phalaenopsis.

Determination of the Antagonistic Effects of Some Rhizospheric Bacteria against Macrophomina phaseolina under In Vitro Conditions

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen causes charcoal rot disease (Sin: Rhizoctonia bataticola) and is responsible for significant yield losses in many plants. In our study, we aimed to evaluate the antagonistic ability of 39 different bacteria, isolated from the fields of sugar beet in 2019, against the pathogen Macrophomina phaseolina isolated from sugar beet, beans and chickpeas. Approximately 31% of the bacteria showed antibiosis effect against the pathogen. It was determined that the effectiveness level of Lelliottia amnigena, Bacillus atrophaeus, B.pumilus and B. cereus (7 isolates) was moderate to high against Macrophomina phaseolina. Bacillus atrophaeus (PTo15-1a) showed the highest efficacy of 80%, 72.94% and 82.35% against Macrophomina phaseolina of chickpea, bean and sugar beet respectively. Lelliottia amnigena (Pto 14-1b) was moderately effective (57.78%) against the chickpea isolate of the pathogen. It was observed that of the seven Bacillus cereus isolates used in the experiment, three isolates (Pto14-1a, Pto12-1b, Pto17-1b) were highly effective against the chickpea pathogen, two (Pto12-1b, Pto14-2b) against bean pathogen, and one (Pto15-1b) against sugar beet isolate. Results have shown varied level of antagonism by different test bacterial against different Macrophomina phaseolina isolates, while the highest level of antibiosis shown by Bacillus atrophaeus against all pathogenic isolates indicated that it can be a potential future bioagent in managing the disease.

Molecular characterization and in vitro control measures of fruit rot disease of Sweet pepper

Fruit rot disease of sweet pepper is one of the main fungal diseases causing huge economic losses to the grower. An experiment was conducted to find out the fungal pathogen associated with fruit rot disease of sweet pepper, obtained from experimental fields of Jahangirnagar University, Bangladesh. Fruit rot disease-causing fungus was isolated from infected fruits and identified using morphological characterization based on colony features, mycelia, conidia as well as molecular characterization based on internal transcribe spacer (ITS) region of the fungus. ITS sequence of our studied fungus MH368146.1 was genetically 99-100% similar to sequences of Fusarium solani in NCBI database. Typical fruit rot symptoms were reproduced by artificial inoculations of the isolated fungus. The mycelial growth of this fungus was evaluated on ten different solid culture media i.e., Potato Dextrose Agar, Yeast Extract Agar, Honey Peptone Agar, Hansen’s Medium, Sabouraud’s Glucose Agar, Kauffman’s Agar, Potato Sucrose Agar, Richard’s Agar and Carrot Agar. Fungus grew well on all tested solid culture media. Several bio-control agents and two commercial fungicides were evaluated against isolated fungus under in vitro condition, in which the highest percent inhibition of radial growth of the fungus was determined as 64.75% due to Trichoderma reesei isolate 2, and 60.63% by Tilt 250 EC (500 ppm) at 7 days post-incubation. Therefore, T. reesei was found as the most suitable to control the growth of F. solani under laboratory conditions. However, further pot and field trials needed to be confirmed the bio-control potential of it. Int. J. Agril. Res. Innov. Tech. 11(2): 108-116, Dec 2021

An Endophytic Strain of Bacillus amyloliquefaciens Suppresses Fusarium oxysporum Infection of Chinese Wolfberry by Altering Its Rhizosphere Bacterial Community

Root rot disease is a serious infection leading to production loss of Chinese wolfberry (Lycium barbarum). This study tested the potential for two bacterial biological control agents, Bacillus amyloliquefaciens HSB1 and FZB42, against five fungal pathogens that frequently cause root rot in Chinese wolfberry. Both HSB1 and FZB42 were found to inhibit fungal mycelial growth, in vitro and in planta, as well as to promote the growth of wolfberry seedlings. In fact, a biocontrol experiment showed efficiency of 100% with at least one treatment involving each biocontrol strain against Fusarium oxysporum. Metagenomic sequencing was used to assess bacterial community shifts in the wolfberry rhizosphere upon introduction of each biocontrol strain. Results showed that HSB1 and FZB42 differentially altered the abundances of different taxa present and positively influenced various functions of inherent wolfberry rhizosphere bacteria. This study highlights the application of biocontrol method in the suppression of fungal pathogens that cause root rot disease in wolfberry, which is useful for agricultural extension agents and commercial growers.

Structure and Function of Rhizosphere Soil and Root Endophytic Microbial Communities Associated With Root Rot of Panax notoginseng

Panax notoginseng (Burk.) F. H. Chen is a Chinese medicinal plant of the Araliaceae family used for the treatment of cardiovascular and cerebrovascular diseases in Asia. P. notoginseng is vulnerable to root rot disease, which reduces the yield of P. notoginseng. In this study, we analyzed the rhizosphere soil and root endophyte microbial communities of P. notoginseng from different geographical locations using high-throughput sequencing. Our results revealed that the P. notoginseng rhizosphere soil microbial community was more diverse than the root endophyte community. Rhodopseudomonas, Actinoplanes, Burkholderia, and Variovorax paradoxus can help P. notoginseng resist the invasion of root rot disease. Ilyonectria mors-panacis, Pseudomonas fluorescens, and Pseudopyrenochaeta lycopersici are pathogenic bacteria of P. notoginseng. The upregulation of amino acid transport and metabolism in the soil would help to resist pathogens and improve the resistance of P. notoginseng. The ABC transporter and gene modulating resistance genes can improve the disease resistance of P. notoginseng, and the increase in the number of GTs (glycosyltransferases) and GHs (glycoside hydrolases) families may be a molecular manifestation of P. notoginseng root rot. In addition, the complete genomes of two Flavobacteriaceae species and one Bacteroides species were obtained. This study demonstrated the microbial and functional diversity in the rhizosphere and root microbial community of P. notoginseng and provided useful information for a better understanding of the microbial community in P. notoginseng root rot. Our results provide insights into the molecular mechanism underlying P. notoginseng root rot and other plant rhizosphere microbial communities.